Caenorhabditis elegans infection model for Coxiella burnetii

 描述(申请人提供)：伯氏柯克斯体是一种传染性极强的细胞内细菌，可引起人类Q热，被归类为选择性病原体。Q热通常表现为一种虚弱的流感样疾病，并伴有肺炎或肝炎， 但在少数病例中，严重的慢性感染与危及生命的心内膜炎 主要的表现形式。人们对柯克斯体的毒力决定因素或细菌如何颠覆宿主细胞知之甚少，尽管这些因素和活动在病原体的生存中发挥了核心作用。我们的总体目标是确定伯氏梭菌新的毒力决定因素，并表征宿主在感染期间对细菌的先天免疫反应。为此，我们最近开发了一种新的秀丽线虫柯克斯体感染的动物模型。当线虫以伯氏线虫为食时，肠道就会持续定植。此外，感染显著缩短了蠕虫的寿命，并产生可见和明显的病理(肛门区畸形或DAR)。以往的报道表明，线虫感染模型中发现的毒力因子在高阶宿主中也得到了类似的利用，并且线虫对病原体的天然免疫反应(IR)的信号通路在高等真核生物中是保守的，这突显了该模型在研究人类病原体方面的价值。我们假设线虫的感染模型可以以高通量的方式用于鉴定柯克斯体的新毒力因子，并分析蠕虫对这种神秘细菌的先天免疫反应的影响因素。为了解决这一假设，目标1将筛选布氏线虫Himar1 TnA7突变体的文库，以降低线虫的适合度，以确定潜在的毒力因素。一旦被确认， 将使用分子科赫假设来研究相应的柯克斯氏菌基因，以验证它们在增强毒力方面的作用。还将进行TNSeq分析，以分析 柯克斯体文库中的每个突变基因在感染过程中都会对细菌的适应性产生影响。我的目标2，我们将通过RNAseq分析线虫的“感染特异性”转录组，以确定针对伯氏线虫的固有IR效应。在目标3中，我们将使用RNAi和线虫突变株的组合来研究这些先天IR效应器的功能，以研究宿主对柯克斯体感染的易感性，相对于野生型线虫。最终，这项研究将通过以下方式显著推动该领域的发展：a)鉴定和分析新的柯克斯体毒力决定因素，b)提供一种新的伯氏柯萨奇杆菌感染的动物模型，这将有助于筛选毒力因子和其他理想的表型，以及c)表征潜在的新的、固有的IR效应，这些效应被宿主用来对抗病原体。

 DESCRIPTION (provided by applicant): Coxiella burnetii is an extremely infectious, intracellular bacterium that causes Q fever in humans and is classified as a select agent. Q fever typically presents as a debilitating, flu-like illness accompanied by pneumonia or hepatitis, but in a minority of cases a severe, chronic infection occurs with life-threatening endocarditis as the predominant manifestation. Little is known about Coxiella's virulence determinants or how the bacterium subverts the host cell, despite the central role for these factors and activities in the pathogen's survival. Our overall goals are to identify novel virulence determinants of C. burnetii and characterize the host's innate immune response against the bacterium during infection. To that end, we recently developed a novel animal model for Coxiella infection in Caenorhabditis elegans. When C. elegans nematodes feed on C. burnetii, the intestines become persistently colonized. Moreover, infection significantly decreases the worm's lifespan and produces a visible and pronounced pathology (deformed anal region or DAR). Previous reports showing that virulence factors identified in the C. elegans infection model are similarly utilized in higher-order hosts and that signaling pathways of C. elegans' innate immune response (IR) against pathogens are conserved in higher eukaryotes, underscores the value of this model for investigating human pathogens. We hypothesize that the C. elegans model of infection can be utilized in a high-throughput fashion to identify novel virulence factors of Coxiella and to analyze effectors of the worm's innate immune response against this enigmatic bacterium. To address the hypothesis, Aim 1 will screen a library of C. burnetii Himar1 TnA7 mutants for reduced fitness in C. elegans to identify potential virulence factors. Once identified, the corresponding Coxiella genes will be investigated using molecular Koch's postulates to verify their role in potentiating virulence. TNSeq will also be done to analyze the contribution of every mutagenized gene in the Coxiella library to bacterial fitness over the course of infection. I Aim 2, we will analyze the "infection-specific" transcriptome of C. elegans by RNASeq to identify innate IR effectors against C. burnetii. In Aim 3, we will investigate the functionality of these innate IR effectors using a combination of RNAi and mutant strains of C. elegans to investigate host susceptibility to a Coxiella infection, relative to wild-type nematodes. In the end, this stud will significantly move the field forward by: a) identification and analysis of novel Coxiella virulence determinants, b) providing a new animal model of C. burnetii infection that will facilitae screening for virulence factors and other desirable phenotypes, and c) characterizing potentially novel, innate IR effectors that are utilized by a host to counter the pathogen.